Our HIV vaccine approach is based on initial immunization with a replicating adenovirus (Ad) vector carrying an HIV gene(s) followed by a booster immunization with an HIV envelope protein. The Ad-HIV vaccine replicates in epithelial cells that line mucosal inductive sites, thus eliciting strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We have shown that initial immunizations with an Ad-HIV vaccine also stimulates production of anti-HIV antibodies. Together, the regimen induces strong and durable protective responses. We have demonstrated that administration of Ad-HIV vaccine to the upper respiratory tract as well as to the gut (by oral immunization) elicits memory T cells that possess "homing receptors" leading them to traffic to the intestine, a prime site of HIV infection. Thus the vaccine approach elicits cellular immunity at a location critical for preventing or controlling HIV infection. Recently we have explored several routes of Ad-recombinant immunization in rhesus macaques including intrarectal, intravaginal, and sublingual as well as the standard administration to the upper respiratory tract intranasally or intratracheally. Interestingly, cellular immune responses as maeasured by interferon-gamma ELISPOT and T cell proliferation assays revealed similar levels of imnmune responses were achieved in peripheral blood by all immunization routes. Administration of an Ad-GFP marker recombinant by the various routes also showed the presence of replicating Ad was similar in rectal tissue and the lung without regard to immunization route. The data suggest use of replicating Ad vectors may facilite vaccine delivery by the easiest, least invasive route. The study of NK cells, key effector cells of innate immunity, is also continuing in order to elucidate their response to Ad-HIV vaccine immunization and their cooperation with vaccine-elicited antibodies in mediating cell killing functions such as antibody-dependent cellular cytotoxicity and antibody-dependent cell mediated viral inhibition. Macaque NK cells have been characterized phenotypically by flow cytometry as a prelude to expanded functional studies. New studies have also shown the importance of CD4 T cell responses in assisting NK cell recall responses. Studies have shown that antibody activities mediated by NK effector cells that span innate and adaptive immunity are correlated with reduced viremia following viral challenge of immunized rhesus macaques. Further, new studies on NKT cells and gamma/delta T cells have recently been initiated to investigate a potential role in vaccine-mediated viremia control. We have also examined Th17 cells in vaccinated versus control macaques to determine if a vaccine which is partially protective could preserve this cell population which is important for control of microbial pathogens. We found that unfortunately, strong viremia control elicited by vaccination does not prevent gradual Th17 cell loss. The implication of this finding is that a vaccine which fully confers sterilizing protection will be necessary to prevent disease sequelae. Finally, we have been exploring use of an immune modulator to facilitate induction of cellular immunity following treatment of macaques with anti-retroviral therapy. A comprehensive picture of responses of various cell types is being compiled and will facilitate further design of both prophylactic and therapeutic vaccine regimens. Overall we have developed expanded capabilities to investigate vaccine-elicited cellular immunity by flow cytometry, as well as by expression of various cytokines and chemokines. Our studies continue to show induction of potent cellular immune responses systemically and mucosally following the replicating Ad-recombinant prime/protein boost regimen.